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RESEARCH SUMMARY

Paramecium is an
excellent model system for studying the biochemical and
electrophysiological mechanisms involved in chemosensory
transductions. Swimming behavior is used in convenient bioassays
to estimate the responsiveness of these free swimming sensory
cells. These unicellular eukaryotic cells can be grown to high
densities in clonal, axenic cultures so that large amounts of
homogeneous cells can be obtained as starting material for
biochemical analyses. Their large size also permits intracellular
voltage clamp analysis and microinjection experiments.
"Genetic dissection" of a sensory transduction pathway
can be approached by selecting for behavioral mutants that have
altered responses to chemoeffectors. Similar studies are also
being pursued in the related ciliate, Tetrahymena. Tetrahymena
have the advantage of higher density growth in a simpler medium
but their small size makes electrophysiological analysis
difficult. Since genomic and cDNA libraries are available for
these ciliates, modern Molecular Biological approaches will be
used to study the regulation and functional expression of the
gene products involved in chemosensory transductions.

SELECTED PROJECTS

Purification, cloning and regulated expression of
and ectonucleotidases in Tetrahymena T. Smith, T. Santangelo, T. Hennessey
Tetrahymena hydrolyze external nucleotide triphosphates
(like ATP and GTP) by an ecto-nTPase and external
nucleotide monophosphates (like AMP and GMP) by a
different ecto-nMPase. The ecto-nTPase may be involved in
inactivating external GTP as a signaling molecule because
GTP is a chemorepellent (Clark et al., 1993). This may be
analogous to a system such as
acetylcholine/acetylcholinesterase. The ecto-nTPase may
also act as either a cell adhesion molecule in the mating
response or in extracellular metabolism of nucleotide
phosphates to provide for their guanine auxotrophy. The
ecto-nMPase may also be involved in this extracellular
scavenge pathway for nucleotides. These two enzymes
(ecto-nTPase and ecto-nMPase) will be characterized in
vivo and in vitro, purified, sequenced, and cloned to
provide the molecular tools necessary to study their
functions and possible regulation of functional surface
expression. Monoclonal antibodies are also being
generated to aid in these efforts.